Molecular cloning and functional characterization of Halloween genes involved in ecdysteroid biosynthesis in the brown planthopper,<em>Nilaparvata lugens</em> (Hemiptera: Delphacidae)
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ACTA ENTOMOLOGICA SINICA  2017, Vol. 60 Issue (10): 1129-1140    DOI: 10.16380/j.kcxb.2017.10.004
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Molecular cloning and functional characterization of Halloween genes involved in ecdysteroid biosynthesis in the brown planthopper,Nilaparvata lugens (Hemiptera: Delphacidae)
LI Kai-Long1,2,*, FU Qiang1, WANG Wei-Xia1, LAI Feng-Xiang1, WAN Pin-Jun1
 (1. State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China; 2. Hunan Provincial Center for Monitoring of Grain Oil Products Quality, Changsha 410201, China)
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Abstract 【Aim】 As one of the most important insects endohormones, ecdysone plays an important role in regulating the development and reproduction of insects. Five Halloween genes are involved in ecdysteroid biosynthesis and encode cytochrome P450 enzymes spook/CYP307A1, Phantom/CYP306A1, disembodied/CYP302A1, shadow/CYP315A1 and shade/CYP314A1, respectively. The brown planthopper, Nilaparvata lugens, is one of the most destructive pests of rice throughout Southeastern and Eastern Asia. For N. lugens, ecdysteroid regulated genes of RNAi technology management has gradually become a research hotspot. The objective of this study is to investigate the function of Halloween genes in N. lugens so as to provide the theoretical basis for the management of N. lugens and development of new pesticides. 【Methods】 Based on the genome and transcriptome database of N. lugens, five ecdysteroidogenesis-related Halloween genes in N. lugens were cloned using RT-PCR, and the deduced protein structure was predicted using different bioinformatics software, and the phylogenetic tree was constructed using the neighbor-joining method of MEGA 5.0. The expression profiles of these genes in different tissues of nymph and at different developmental stages of N. lugens were detected by quantitative real-time PCR. RNAi experiment was carried out to examine the effects of knockdown of Halloween genes on the development and reproduction of N. lugens. 【Results】 Five Halloween genes involved in ecdysteroidogenesis, i.e., NlCyp307a1, NlCyp306a1, NlCyp302a1, NlCyp315a1, and NlCyp314a1, were cloned and identified in N. lugens. Phylogenetic analysis revealed that the five Halloween genes belong to two superfamilies 2-Clan and Mito-Clan, respectively, and clustered with their homologous genes in other insects, suggesting that Halloween genes and edysteroidogenesis pathways are very highly conserved during insect evolution. NlCYP314A1 contains five conserved P450s motifs and two transmembrane domains, being a typical mitochondria cytochrome P450s. The results of qRT-PCR showed that five Halloween genes had higher mRNA abundance at 24 h and 60 h after molting in the 5th instar nymph stage. NlCyp314a1 was continually expressed during the whole developmental stage, and showed the highest expression level in adults. Among the examined tissues, thorax had the higher transcription level of NlCyp314a1 than head and abdomen, and fat body had the highest transcription level of NlCyp314a1, followed by leg, integument, wing bud, and midgut. Four days after injection of dsNlCyp314a1, the expression levels of NlCyp314a1 and NlFTZ-F1 in the treated individuals were extremely significantly decreased by 84.6% and 64.1%, respectively, compared with the control. NlCyp314a1 knockdown resulted in nymphs failed to molt on time, exhibited apparent phenotypic defects, and >95% mortality of the nymphs at day 7 after injection. NlCyp314a1 knockdown also resulted in the developmental abnormality of adult ovary. 【Conclusion】 Halloween genes of N. lugens involving in ecdysteroidogenesis are evolutionarily conserved. Silencing of selected Halloween gene expression causes phenotypic defects in molting and reproduction. These results suggest that Halloween genes encode functional proteins that are involved in molting and reproduction of N. lugens. Halloween genes might be used in altering genetically the progress of molting and reproduction of N. lugens. This study provides a theoretical basis for potential application of Halloween genes in the management of N. lugens.
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LI Kai-Long
FU Qiang
WANG Wei-Xia
LAI Feng-Xiang
WAN Pin-Jun
Key wordsNilaparvata lugens; ecdysone   Halloween gene   gene expression   molting   reproduction     
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LI Kai-Long,FU Qiang,WANG Wei-Xia et al. Molecular cloning and functional characterization of Halloween genes involved in ecdysteroid biosynthesis in the brown planthopper,Nilaparvata lugens (Hemiptera: Delphacidae)[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(10): 1129-1140.
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